This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacillus anthracis spores can cause significant morbidity and mortality if released into the environment. There is a need for both an effective vaccine as well as targeted immunotherapeutic agents. The currently licensed vaccine requires six vaccinations, annual boosters, and has been associated with serious adverse events. Thus there has been a major effort made to develop an improved vaccination strategy which can generate lasting protective immunity with reduced vaccinations. Dr. James'laboratory has been working to identify the major humoral targets of B. anthracis that provide protection. Human monoclonal antibodies were previously generated from individuals who received the anthrax vaccine. Each of these monoclonal antibodies has previously been characterized in both in vitro and in vivo toxin neutralization assays as well as by linear epitope and protease fragment mapping studies. Multiple functionally informative anti-PA monoclonal antibodies were identified. However, the structural epitopes recognized by these functionally informative monoclonal antibodies remained unclear. These monoclonal antibodies apparently recognized conformational epitopes rather than the linear peptide epitopes of protective antigen. The goal of this study was to define the protective antigen structural determinants that were specifically recognized by the functionally informative monoclonal anti-PA antibodies. Using the three dimensional structure of anthrax PA as a guide, different domains of PA will be cloned as recombinant fusion proteins. These PA subdomain containing fusion proteins will then be expressed and used in protein binding assays to define which PA domains are required for anti-PA monoclonal antibody binding. The results of these studies will identify which domains of PA are the best targets for generating protective immune responses through vaccination.